【作者】 郭先武；

【导师】 陈华癸；

【作者基本信息】 华中农业大学 ， 微生物学， 1999， 博士

【摘要】 根瘤菌群体遗传学的比较研究对象主要集中在以下几个方面：来自不同地区同种植物根瘤菌菌株、同一地区的不同菌株、有宿主植物分布与长期没有宿主植物分布的土著性根瘤菌的群体遗传学比较，但由于从相同植物中分离出来的根瘤菌并不一定是同种根瘤菌，从而对群体的比较研究带来了许多困难。另一方面，由于群体的研究要处理大量的样本，难于深入细致地对每一个菌株作较为充分的研究，许多有规律的东西也不一定反映出来。华癸根瘤菌(Mesorhizobium huakuii)在这方面有得天独厚的优势，研究表明其专一宿主植物紫云英系独立的互接种族，主要分布在我国南方，近年来华癸根瘤菌的分子遗传学研究又有许多新的进展，因此，本研究选用华癸根瘤菌为研究对象，以采自我国南方7省的紫云英植株上直接分离的208个菌株进行群体遗传学特性的研究，并将质粒上的基因与染色体上基因的群体特性进行比较，以从群体的角度认识两者之间的关系，对华癸根瘤菌的进化有进一步的了解。 对染色体基因的认识是从16S rDNA的研究入手。用pA和pH这对引物，对所有供试菌株进行PCR扩增，其产物均为1条1.5kb片段，用识别四个碱基的4种限制性内切酶进行RFLP分析，共发现四种16S rDNA基因型，其中a、b两型之和约占供试菌株的95.2％。 选取其中三种类型的四个代表菌株(HB5A4-1、HN14A16-2、ZJ15B7-1和HN8A1-1)进行部分序列测定(600bp-800bp)。通过与其它标准菌株遗传距离的比较发现，它们均属于Mesorhizobium，并与华癸根瘤菌的标准菌株M. huakuii CCBAU2609有紧密的亲缘关系。故应属于华癸根瘤菌。但种内菌株间表现出很大的遗传差异。165 rDNA序列的差异与其表型差异没有明显的一致性。 为了更详细地考察染色体遗传信息在菌株之间的差异，选用165一235rDNA基因l司序列(IGS)作进一步的研究。用pHr和p23SR01为引物扩增出不同大小的PCR产物，通常为1 .8一2.okb(含235 rDNAI.okb)，实际的IGS长度为0.8一1 .okb。但有少数菌株产生54obP的DNA片段，菌株ZJSA16一1则同时产生两种不同大小片段。产生540bP扩增产物的菌株表明它们还存在235 rDNA的重组型结构。对所有菌株的IGS进行盯LP分析发现了14种IGS酶切类型，其中IGS一1、2、3是主要代表，共占总菌株的85%，其它类型相对极少。这些不同的IGS类型的菌株有一定地域分布特点，也表现与165:DNA的密切对应关系，只有十多个菌株例外。对这些菌株IGS酶切类型的聚类分析表明，在90%同源水平上可以分成5群，每一个群体内均有一种占优势的基本类型。在80%的同源水平上可以分成三大群，其分布的地域性特点更加明显。 质粒是根瘤菌遗传物质的重要组成部分，含有重要的遗传信息，华癸根瘤菌的共生基因主要位于其上。选用101个菌株进行质粒类型的分析，发现了16种不同的质粒类型，所含质粒1一4个不等，其分子量在36-364MD之间。分子杂交结果证明，这些菌株均含有共生质粒(第一或第二大质粒)，其大小均在looMD以上。由于质粒型在特定的菌株中是相对稳定的，同一土壤中质粒类型的多样性应为进化的结果，即特定的质粒型是菌株的特定染色体背景与细菌环境相互作用的结果。7653R菌株共生质粒的luxAB基因标记为进一步的分子生态学研究提供了材料，还同时发现质粒和染色体片断在特定环境条件下也会发生缺失。7653R质粒缺失菌株的结瘤实验表明即使共生质粒缺少1/3，依然具有结瘤能力，可见其共生质粒上至少有1/3的片段与形成共生关系无关。 在自然群体中，共生基因的分布状况又是怎样的呢?对此进行探针杂交的盯LP分析。用Eco刀酶切总DNA，以含nodDBC的7653R 4.2kb片段为探针杂交，发现了9种不同类型。由于在208个供试菌株中，1条阳性带的占绝大多数，且都比4.2kb大，因此，认为这个杂交的结果主要反映的是4.2kb以外的DNA片段酶切位置的变化。与染色体基因型比较发现，二者有很强的对应关系，说明二者间存在协同进化。以Pstl酶切总DNA用同样的探针杂交，反映出丰富的杂交信号，通过聚类分析及与染色体基因型的比较得出:即染色体基因型及共生基因型不表现亲缘关系的一致性，反映二者起源可能有别或进化过程中发生了重组。根据Pstl的酶切杂交结果可以看出一条属于4.2kb内部2.Okb片段的非常保守，这个2，okb片段应含nodB及部分nodD.C。为了进一步考察该片段在不同菌株中的变异，特设计一对引物，进行PCR一即LP分析，发现所有26个代表性菌株中的共生基因均有极高同源性，其变异性与染色体基因型无关。不同染色体基因型菌株有着共同结瘤基因的相同变异只能说明在进化的历程中有共生质粒DNA水平转移发生，这是证明质粒进化过程中存在共生质粒DNA水平转移的有力证据。 通过以上分析提出了共生基因及根瘤菌进化的基本思想:①根瘤菌在进化过程中发生了共生基因的丢失，转移及重组。②共生质粒上共生功能区与宿主植物、非功能区与细菌染色体之间均存在协同进化。它解释了共生基因与165 rRNA在划分物种亲缘关系上的不一致性、某些根瘤菌与非共生细菌比它与更多还原

【Abstract】 208 isolates from Astragalus sinicus were collected from 51 locations, 7 provinces in southern China. 16S rRNA gene and 16S-23S rDNA intergenic spacer are the representation of chromosomal information and common nod genes (nodD,B,C) as the marks of pSym information.All the strains produced only one 1.5kb fragment of 16S rDNA by PCR-amplification with the primes pA and pH. With the respective disposition of restriction endonucleases Hinfl. Mspl. Haelll and TaqI, four types of band pattens were presented. The 16S rDNA of four strains representing three types of strains were partially sequenenced. By comparing with the related sequeane of other rhizobia, the results from clustering analysis indicated that the four strains are all belonged to Mesorizobium but in two groups, HB5A4-1 and HN14A16-2 choser to M. loti, ZJ15B7-1 and HN8A1-1 more related to M. huakuii, CCBAU2609. The great discrepancy among the strains is present in M. huakuii.Using the primes, PHr and p23SR01, for amplifying 16S-23S intergenic spacer (IGS), 202 isolates produced one fragment, 1.8-2.0 kb, and 5 isolates produced unique land, 540bp, except one isolate with both the fragments, The result suggests that the recombination of rDNA operon occurred in evolution. By the digestion with the same restriction endonucleases as above, 14 IGS-types were produced, which can by placed into 5 groups (90% similarity level). In contrast to 16S rDNA types, these IGS-types are predominantly in conformity with them.101 strains isolated from various locations were examined by the Echardt’s method and produced 16 types of plasmid profiles, 1-4 plasmids and 36-360 MDa in size. The hybridization of plasmid DNA, with the probe, nodDBC (4.2kb) cloned from M.huakuii 7653R, indicated that the nodulation genes are all located on the largest or the second plasmids.EcoRl-digested genomic DNAs of all the isolates are hybridized with nodDBC probe and nine types of positive band patterns were discovered. There are strong correspondences between the chromosomal genotypes and the hybriding types, indicatingthat coadaptation between them may occur. By the hybridization of Pst-I digested genomic DNA with the same probe, 28 strains including all the EcoRI-digested hybriding types of isolates created 10 types of hybridization patterns and their dendrogram was constructed. In comparison of the chromosomal genotypes. There is no closer relationship among their plasmid genotypes in certain chromosomal background and vice versa. It implies that the origin of common nod genes may be different from that of chromosomal genes.We noticed that a conservative fragment of 2.0kb occurs in almost all the strains by analyzing the positive bands in the Pstl-hybriding pattern. It should be the sequence including nodB and partial nodD and C according to corresponding sequence of M. huakuii 7653R. In order to examine the variation of the fragment in the isolates. A pair of primes, pG1 and pG2 was designed for its PCR-amplification. 25 strains in various chromosomal and plasmid genotypes all produced the expecting fragment. By the digestion with HindIIL MspK HinfI and HaeIII, only the disposition with HaeIII gave different types, one type involved in 20 strains and two mutational types in other strains. It is very interesting that there is the same mutational type in different chromosomal background. It confirms that gene transfer had taken place in its evolution and furthermore, the mutation happened before gene transfer.Finally, we profound one hypothesis: (1) the loss, recombination of symbiotic genes and gene transfer exist in the evolutional process of rhizobia. (2)there is the coadaptation of not only between symbiotic sequences of rhizobia and its host plant but also between the nonsymbiotic sequences and chromosomal genes and chromosomal genes.更多还原